Computational protocol: Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus

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Protocol publication

[…] The total RNA of each sample was extracted using TRIzol reagent (Invitrogen, USA). RNA quality and quantity were evaluated using Agilent Bioanalyzer 2100 (Palo Alto, USA) and NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), respectively. RNA samples with integrity number (RIN)>8.0, 28S/18S>1.4 and OD260/280>1.8 were used for library preparation. Three cDNA libraries were generated using NEBNext® Ultra RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions. Transcriptome sequencing was performed using Illumina HiSeqTM 4000 platform that generated 150 bp paired-end raw reads. All sequencing data were deposited in the NCBI Sequence Read Archive ( under the accession number SRP127013.Raw reads were filtered with Trimmomatic (ver 0.30) [] to remove adaptor contamination, unqualified reads and sequences. High quality clean reads were pooled and de novo assembled using Trinity [] with default parameters, forming transcripts. Then the TIGR Gene Indices clustering tools (TGICL) (ver 2.1) [] were used to cluster and remove redundant transcripts, and identify unigenes (i.e., non-redundant transcripts), which were used for subsequent analysis. […]

Pipeline specifications

Software tools Trimmomatic, Trinity, TGICL
Databases SRA
Applications RNA-seq analysis, Transcription analysis
Organisms Misgurnus anguillicaudatus